Monoclonal antibody stability represents a current challenge in the purification and formulation of these proteins. MAb instability leads to high levels of aggregated mAb in protein formulations, which can have several disadvantages including changing protein activity and potentially leading to undesirable immunological responses in patients. Protein A affinity chromatography is a powerful and widely-used tool for purifying antibodies. In order to elute a protein or antibody from the Protein A resin, acidic conditions are required due to the high affinity of the monoclonal antibodies to the resin. Exposure to these acidic conditions can result in the formation of protein aggregates. Some strategies to address aggregation during Protein A chromatography have been previously described in the literature. Furthermore, a low pH hold step following elution is required for viral inactivation and can also result in the formation of protein aggregates.
Furthermore, association and aggregation tend to occur during frequently. Extensive research into changes in antibody structure caused by acidic pH has been conducted. However, resolution of the issues regarding structural change and the association and aggregation reactions has yet to be proposed.
The performance of the cell culture process can have significant effects on product quality and potency, especially with respect to glycosylation, post-transcriptional modifications and impurity profiles. Since CHO cell and other continuously cultured cells have low efficiency in completely oxidizing glucose to CO2 and H2O, one by-product of cell culture process is lactate accumulation, which can cause acidification of culture medium and lead to high osmolality and low viability due to the alkali added to control the medium pH. Thus, when lactate accumulation exceeds the buffering capacity of the culture medium, pH drifts downward, which could trigger base addition leading to increased osmolality of the culture medium. This could be risky in cell lines that synthesize excessive amounts of lactate since high pH, high lactate and high osmolality cascade often causes delayed cell growth and accelerated cell death.
The impact of osmolality has been reported on growth inhibition with increasing osmolality and effect on cell specific productivity (deZengotita V M, Schmelzer A E, Miller W M. Characterization of hybridoma cell responses to elevated pCO2 and osmolality: intracellular pH, cell size, apoptosis and metabolism. Biotechnol Bioeng. 2002; 77:369-380). These deleterious effects could be exacerbated when combined with high dissolved CO2 levels that could occur in high cell density cultures, and hence it is vital to ensure during process development that the osmolality profile is acceptably low, especially towards the latter stages of the cell culture process. (Zhu M M, Goyal A, Rank D L, Gupta S K, Boom T V, Lee S S. Effects of elevated pCO2 and osmolality on growth of CHO cells and production of antibody-fusion protein B1: A case study. Biotechnol Prog. 2005; 21:70-77). Also it has been reported earlier that when high feeding rates are utilized both lactate and ammonium start accumulating at higher concentrations in the cultures resulting in an osmolality as high as 500 mOsm/kg to 700 mOsm/kg.